OptoPI3K, genetic code expansion, and click chemistry reveal mechanisms underlying reciprocal regulation between TRPV1 and PI3K

Reviewer #1 (Public Review):

Summary:
This work seeks to isolate the specific effects of phosphoinositide 3-kinase (PI3K) on the trafficking of the ion channel TRPV1, distinct from other receptor tyrosine kinase-activated effectors. It builds on earlier studies by the same group (Stein et al. 2006; Stratiievska et al. 2018), which described the regulatory relationship between PI3K, nerve growth factor (NGF), and TRPV1 trafficking. A central theme of this study is the development of methods that precisely measure the influence of PI3K on TRPV1 trafficking and vice versa. The authors employ a range of innovative methodologies to explore the dynamics between TRPV1 and PI3K trafficking.

Strengths:
A major strength of this study is the application of innovative methods to understand the interaction between PI3K and TRPV1 trafficking. The key techniques presented include:

1. The optogenetic trafficking system based on phytochrome B, introduced in this research. Its interaction mechanism, dependent on reversible light activation, is comprehensively explained in Figures 1 and 2, with the system's efficacy demonstrated in Figure 3.

2. An extracellular labeling method using click chemistry, which although not exclusive to this study, introduces specific reagents engineered for membrane impermeability.

The central biological insight presented here is the sufficiency of PI3K activation to guide TRPV1 trafficking to the plasma membrane. An additional notable discovery is the potential regulation of insulin receptors via this mechanism.

The paper's strengths are anchored in its innovative methodologies and the valuable collaboration between groups specializing in distinct areas of research.

Weaknesses:
The paper might benefit from a more streamlined structure and a clearer emphasis on its findings. A possible way to enhance its impact might be to focus more on its methodological aspects. The methodological facets stand out as both innovative and impactful. These experiments are well-executed and align with biological expectations. It's evident how these techniques could be tailored for many protein trafficking studies, a sentiment echoed in the manuscript (lines 287-288). When seen through a purely biological lens, some findings, like those concerning the PI3K-TRPV1 interaction, are very similar to previous work (Stratiievska et al. 2018). A biological focus demands further characterization of this interaction through mutagenesis. Also, the incorporation of insights on the insulin receptor feels somewhat tangential. A cohesive approach could be to reshape the manuscript with a primary focus on methodology, using TRPV1 and InsR as illustrative examples.

Reviewer #2 (Public Review):

Summary:
The authors hypothesized that the interaction between TRPV1 and PI3K directly influenced PI3K activity along with increasing TRPV1 trafficking to the membrane. Previous results showed that PI3K could interact with one of the ankyrin repeat domains, however it was unclear whether the direct interaction influenced PI3K activity.

Strengths:
A major strength of the paper is the innovative combination of techniques. The first technique used the optogenetic PhyB/PIF system. They anchored PhyB to the membrane and fused PIF with the interSH2 domain from PI3K. This allowed them to use 650nm light to induce an interaction between the PhyB and PIF resulting in a recruitment of the endogenous PI3K to the membrane through the iSH2 domain without actual activation of an RTK. This allowed them to dissect out one function, just PI3K recruitment/activation from the vast number of RTK downstream cascades.

The second technique was the development of a new non-canonical amino acid that is cell-impermeant. The authors synthesized the sTSO-sulfa-Cy5 compound that will react with the Tet3 ncAA through click chemistry. They showed that the sulfa-Cy5 did not cross the membrane and would be used to track protein production over time, though the reaction rates were slow as noted by the authors. The comparison of the sulfa-Cy5 data with the standard GFP with TIRF showed a clear difference indicating the useful information that is gained with the ncAA.

Weaknesses:
To monitor the phosphatidylinositol-3,4,5-trisphosphates, the pleckstrin homology (PH) domain from Akt was used. This PH domain is not specific for just PI(3,4,5)P3 as stated by the authors. The Akt PH domain also binds PI(3,4)P2. The observed PI3K localization increase will also increase PI(3,4)P2 concentrations so the observed responses may not be solely because of PI(3,4,5)P3.

The data in Figure 4 supplement was confusing to interpret since it is unclear whether a membrane protein with the Tet3 is being expressed at the same time as the ncAA for labeling or if the observed labeling is endogenous. If the observed labeling in Figure 4 supplement D is endogenous, then significant concerns come up regarding the background labeling of the sTCO-sulfo-Cy5 used in the rest of the experiments.

Even with the weaknesses, I believe the authors did achieve their goal of investigating the reciprocity between TRPV1 and PI3K. Their results support their conclusions and will help understand how TRPV1 is regulated by signals other than the traditional channel activators. The tools developed in the article will be of use to the broader cell biology and biophysics community, not just the channel community. The opto control of the PhyB/PIF system makes it more convenient than other systems since it does not take the typical wavelengths needed for fluorescence. The cell-impermeant ncAA will also be a great tool for those studying membrane proteins, protein trafficking and protein dynamics.

Reviewer #3 (Public Review):

Summary: In this manuscript, Koh, Stratiievska, and their colleagues investigate the mechanism by which TRPV1 channels are delivered to the plasma membrane following the activation of receptor tyrosine kinases, specifically focusing on the NGF receptor. They demonstrate that the activation of the NGF receptor's PI3K pathway alone is sufficient to increase the levels of TRPV1 at the plasma membrane.

Strengths: The authors employ cutting-edge optogenetic, imaging, and chemical-biology techniques to achieve their research goals. They ingeniously use optogenetics to selectively activate the PI3K pathway without affecting other NGF pathways. Additionally, they develop a novel, membrane-impermeable fluorescent probe for labeling cell-surface proteins through click-chemistry.

Weaknesses: Previous research, including work by the authors themselves, has already established that PI3K activation is required for NGF-induced TRPV1 trafficking to the plasma membrane. Moreover, the paper suffers from issues such as subpar writing quality, a lack of statistical analysis, and insufficient control experiments, which dampen the reviewer's enthusiasm.